Phosphate uptake by absorptive epithelia has been demonstrated to occur by a pH-sensitive, Na-dependent mechanism. The uptake process has been localized to the brush border membrane of intestinal cells where an arsenate-sensitive Na-dependent cotransport mechanism similar to that described for sugars and amino acids has been reported. It is the immediate goal of this proposal to isolate the Na/phosphate cotransporter and determine its structure. The long-term goal is to examine regulation of the cotransporter by vitamin D and dietary phosphate. The specific aims of this project are to: 1) isolate and purify the functional cotransporter to homogeneity; 2) determine the structure of the cotransporter (sugar content, monomer/dimer, molecular mass, pI, amino acid composition, and amino acid sequence of substrate sites); 3) determine the molecular topography of the protein and active sites (fluorescent quenching of substrate site probes, fluorescence energy transfer, tryptophan and tyrosine fluorescence); 4) examine the cotransporter conformational changes (proteolytic digestion, tryptophan energy transfer and quench reagents, and substrate-site quenching; and 5) examine the effect of vitamin D and dietary phosphate on the number of membrane expressed cotransporters and cytoplasmic cotransporters. These studies will have a general impact on our understanding of the mechanism of sugar, amino acid, and metabolic intermediate transport across plasma membranes of the kidney, intestine, liver, and brain. These studies offer a unique opportunity to examine hormonal regulation of a transport protein.